There are 3 TGF-beta isoforms present in humans, TGF-beta 1, TGF-beta 2 and TGF-beta 3. The isoforms are homologous and share ˜70% sequence identity. They are all synthesised and secreted as a latent complex in which TGF-beta is complexed with two other polypeptides, latent TGF-beta binding protein (LTBP) and latency-associated peptide (LAP) (a protein derived from the N-terminal region of the TGF-beta gene product). Serum proteinases such as plasmin catalyze the release of active mature TGF-beta from the complex.
In their active forms, TGF-beta isoforms exist as a ˜25 KDa homodimeric protein. All 3 isoforms signal via the same transmembrane receptors TbetaRI and TbetaRII. TGF-beta first binds to TbetaRII which then forms a heterotetrameric complex with TbetaRI, leading to phosphorylation of TbetaRI and activation of subsequent signalling pathways (see Derynck & Miyazono (eds), 2008, The TGF-beta Family, Cold Spring Harbor Press). Despite signalling via the same receptor complex, distinct non-overlapping functions of the 3 isoforms have been noted which is exemplified by mice containing genetic deletions of the individual isoforms each having different phenotypes (Shull et al., 1992, Nature 359: 693-699; Sanford et al., 1997, Development 124: 2659-2670; Proetzel et al., 1995, Nature Genet., 11: 409-414).
TGF-beta is a pleotropic molecule involved in a range of biological processes. TGF-beta inhibits the proliferation of many cell types, including epithelial, endothelial, haematopoietic and immune cells. The effector functions of immune cells are also responsive to TGF-beta and TGF-beta suppresses Th1 and Th2 cell differentiation whilst stimulating Treg cells, thus TGF-beta has a predominantly immunosuppressive function (Li et al., 2006, Ann Rev Immunol., 24: 99-146; Rubtsov & Rudensky, 2007, Nat Rev Immunol., 7: 443-453). TGF-beta expression is highly regulated and involved in maintenance of tissue homeostasis. However chronic over expression of TGF-beta is linked with driving disease progression in disease states such as cancer and fibrosis.
Due to the role of human TGF-beta in a variety of human disorders, therapeutic strategies have been designed to inhibit or counteract TGF-beta activity. In particular, antibodies that bind to, and neutralize, TGF-beta have been sought as a means to inhibit TGF-beta activity. Antibodies to TGF-beta are known in the art. A systemically administered anti-TGF-beta1 antibody (CAT-192) was evaluated in a Phase I/II trial in systemic sclerosis patients, with no evidence of efficacy with doses up to 10 mg/kg (Denton et al., 2007, Arthritis Rheum, 56: 323-333). A humanised antibody (TbetaM1) optimised for activity against TGF-beta1 was assessed in a Phase1 trial in patients with metastatic cancer, but no anti-tumor effect was noted (Cohn et al., 2014, Int J Oncol., 45: 2221-2231). A human TGF-beta2 antibody (CAT-152) was evaluated for prevention of scarring after trabeculectomy, but no difference from placebo was noted (CAT-152 0102 Trabeculectomy Study Group, 2007, Ophthalmology, 114: 1822-1830). A systemically administered full length IgG specific for TGF-beta1, 2 and 3 (Fresolimumab, GC1008) has been investigated for the treatment of certain cancers and fibrotic disease. However, side effects have been reported including skin lesions that appear to be associated with systemic delivery of the antibody (Lacouture et al., 2015, Cancer Immunol Immunother., 64: 437-446).
Fibrosis is an aberrant response to wound healing wherein excess fibrous connective tissue is formed in an organ or tissue. In the remodelling phase during normal wound healing, synthesis of new collagen exceeds the rate at which it is degraded, resulting in scar formation. The final process of normal wound healing is scar resolution which occurs through a combination of reduced collagen synthesis and increased collagen degradation, a process controlled by matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPS) produced by granulocytes, macrophages, epidermal cells and myofibroblasts. Thus wound healing involves a shift in metabolic equilibrium from stimulation of deposition followed by resolution. Any disruption in this equilibrium may result in excessive deposition of matrix components resulting in hardening and scarring of tissues and destruction of normal tissue architecture and a compromise in tissue function; this disruption is termed fibrosis.
Abnormal epithelial-mesenchymal interactions, altered fibroblast phenotypes, exaggerated fibroblast proliferation, and excessive deposition of collagen and extracellular matrix are all the key processes which contribute to fibrotic disease. A key cell type in this process is the myofibroblast. Activation of myofibroblasts results in their increased secretion of types I, III and IV collagen, fibronectin, laminin and proteoglycans. Other cell types considered to play a prominent role in fibrosis include epithelial cells and macrophages. TGF-beta is considered to be a master regulator of fibrosis and contributes to the fibrotic process via actions on several cell types including macrophages and fibroblasts (Leask & Abraham, 2004, FASEB J., 18: 816-827). Key profibrotic activities include the stimulation of fibroblast migration and the transformation of fibroblasts to myofibroblasts, stimulating excessive ECM deposition. TGF-beta is also involved in macrophage migration and stimulates the production of mesenchymal growth factors from macrophages such as PDGF, as well as inhibiting ECM degradation through the increased expression of protease inhibitors such as TIMP3.
Fibrotic diseases are a leading cause of morbidity and mortality and can affect many tissue and organ systems. Included in this group of diseases are interstitial lung diseases. Idiopathic pulmonary fibrosis (IPF) is the most common form of interstitial lung diseases and is one of seven distinct groups of idiopathic interstitial pneumonias (IIP). The interstitium is the microscopic space between the basement membranes of the alveolar epithelium and capillary endothelium, and forms part of the blood-gas barrier. IIPs are characterised by expansion of the interstitial compartment by inflammatory cells, with associated fibrosis particularly noted for IPF.
IPF patients present with progressive exertional dyspnoea and cough with progressive pulmonary parenchymal fibrosis, resulting in pulmonary restriction and hypoxemia. The diagnosis of IPF is established using a combination of clinical, radiographic and pathological criteria and is associated with a characteristic pathological pattern called usual interstitial pneumonia (UIP).
IPF can be diagnosed at any age, but is most prevalent in those aged over 50 years and prevalence is higher in men than women. IPF has a mortality rate higher than many neoplastic diseases, with a 3 year survival rate of 50% and a 5 year survival rate of only 20%. The cause of IPF is unknown, but it is hypothesised that there are multiple episodes of epithelial cell activation from as yet unidentified exogenous and endogenous stimuli, which if left untreated leads to progressive lung injury and ultimately fibrosis. Disruption of the alveolar epithelium is followed by migration, proliferation and activation of mesenchymal cells, resulting in the formation of fibroblastic/myofibroblastic foci with excessive accumulation of ECM.
TGF-beta expression is increased in the fibrotic lungs of IPF patients (Broekelmann et al., 1991, PNAS, 88: 6642-6646; Khalil et al., 1991, Am J Respir Cell Mol Biol, 5: 155-162) and together with the well-established role of TGF-beta in driving fibrotic mechanisms the inhibition of TGF-beta should be considered as an effective mechanism for the treatment of IPF patients.
There is no effective therapy available for IPF patients. Anti-inflammatory agents, including corticosteroids, cyclophosphamide and azothiaprine have proved to be of little benefit for patients and have associated side effects. Recently two small molecule drugs, pirfenidone and nintedanib, have been approved for the treatment of IPF. Both drugs have been shown to slow the progression of disease, but neither cures the disease and many patients continue to decline. In addition treatment-related adverse events such as gastrointestinal events, rash and photosensitivity are evident (Cottin and Maher, 2015, Eur Respir Rev, 24: 58-64; Mazzei et al., 2015, Ther Adv Respir Dis.) To date, no targeted therapies and no antibody therapies have been approved for fibrotic indications.
Furthermore, TGF-beta is also associated with pulmonary hypertension, such as pulmonary arterial hypertension (PAH). Increased expression of TGF-beta in patients with pulmonary hypertension has been shown by immunohistochemistry (Botney et al., 1994, Am J Pathol, 144: 286-295) and also noted in blood and lung homogenates from pulmonary hypertension patients (Selimovic et al., 2009, Eur Respir J, 34: 662-668; Gore et al., PLOS One (2014) 9(6):e100310). A TbetaRI kinase inhibitor has also been shown to inhibit the monocrotaline-induced model of pulmonary hypertension (Zaiman et al., 2008, Am J Respir Crit Care Med, 177: 896-905). Pulmonary hypertension is a well-recognised complication of IPF, and these data support the hypothesis that IPF patients whose symptoms are driven by both interstitial fibrosis and pulmonary hypertension could be a sub-population of patients for whom anti-TGF-beta therapies could potentially be even more effective.
Therefore, there exists a need in the art for suitable and/or improved antibodies capable of binding and inhibiting all three isoforms of TGF-beta suitable for therapeutic applications. Such antibodies may also be more effective for treating pulmonary indications and/or have fewer side effects if delivered by inhalation.